The proposed research project seeks to investigate the mechanisms by which cells polarize and help further our understanding of the role of polarization in directed cell migration. These studies will use the social amoeba Dictyostelium discoideum and will be conducted using a newly developed open microfluidic device made out of Polydimethylsiloxane. This device has multiple, micron-sized channels which can be used to race morphologically distinct cells against each other and the channels are also capable of confining cells for polarity studies. By learning more about D. discoideum cells, these findings can be correlated with the chemotaxis processes controlling other eukaryotic cells such as neutrophils and also elucidate the pathways that regulate cancer metastasis. In order to achieve these goals, three specific aims have been set. The first specific aim focuses on the further development of an open microfluidic devices that can be used for chemotaxis assays, and allows the visualization of a stable, passive chemical gradient. The second specific aim targets the role and mechanisms of polarity in chemotaxing cells by comparing the migration of polarized and unpolarized D. Discoideum cells towards their respective chemoattractants. Cells expressing fluorescent cytoskeletal and signaling proteins will be exposed to chemoattractant at one end of a microfluidic channel, after which the gradient will be reversed and the redistribution of these fluorescent molecules will be analyzed as the cells de-polarize and re-polarize. The third specific aim focuses on the gradient sensing mechanism in eukaryotic cells. The concentration of the gradient (input) will be compared to the migration speeds and signaling protein localizations (output). In line with the mission of the National Institute of General Medical Sciences (NIGMS), this research proposal seeks to understand the chemotaxis processes of eukaryotic cells. The findings from these experiments will help elucidate the basic mechanisms by which cells sense chemical gradients and migrate and further our understanding of how cancer cells metastasize. The proposed research program will require the P.I. to apply theory learned in both the classroom and from scientific literature. Appropriate research techniques will be picked up in the laboratory and the experiments will be performed under the guidance of the research sponsor. Training and implementation on the use of appropriate equipment and techniques will be facilitated by qualified personnel in the Janetopoulos laboratory and the staff at Vanderbilt University medical center.